PROJECT SUMMARY/ABSTRACT Cardiovascular disease (CVD) is the leading cause of death in the United States. Atherosclerosis, the major cause of CVD, is an inflammatory disease resulting from the accumulation of cholesterol in plaque along the artery walls. High density lipoprotein (HDL) protects against CVD, as it carries peripheral cholesterol to the liver for biliary excretion in a process known as reverse cholesterol transport (RCT). Efficient RCT and the selective uptake of cholesteryl esters (CE) from HDL into cells require interactions between HDL and its high affinity receptor, scavenger receptor-BI (SR-BI). The presence of SR-BI dimers and higher-order oligomers is important for facilitating the selective uptake of HDL-CE; however, the mechanisms that drive oligomerization remain unknown. Both the N-terminal and C-terminal transmembrane domains (N-TMD and C-TMD, respectively) of SR-BI contain putative dimerization motifs that, when disrupted, impair SR-BI dimerization as well as cholesterol transport functions. Recently, our lab used NMR techniques to solve the high-resolution 3D structure of a peptide of SR-BI (residues 405-475) that spans the C-TMD, as well as a leucine zipper dimerization motif. Using this structure to our full advantage, we have designed experiments in this fellowship application to test the hypothesis that SR-BI oligomerization and flexibility between the transmembrane domains of neighboring SR-BI monomers are essential for efficient RCT. In Aim 1, we will test the importance of key structural features of SR-BI?s C-TMD on cholesterol transport functions of SR-BI. In Aim 1.1, we will study the significance of a proline kink within the C-TMD, while in Aim 1.2, we will examine the effects of reduced flexibility between C-TMDs of SR-BI monomers using a ?locked dimer? approach. In Aim 1.3, we will determine the physiological relevance of oligomerization by testing the leucine zipper SR-BI mutant in an in vivo model of RCT. In Aim 2, we will determine the NMR structure of SR-BI?s N-TMD in order to identify structural features of this domain that may be functionally relevant (Aim 2.1). Then, armed with NMR structures of both transmembrane domains, we will map the dimer interface(s) that support SR-BI oligomerization between the C-TMDs and/or N-TMDs using paramagnetic relaxation enhancement strategies in Aim 2.2. These studies will help us to better understand how SR-BI/HDL interactions, together with SR-BI oligomerization, promote efficient RCT and improve net cholesterol excretion. Our findings will hopefully lead to the design of a new class of therapeutics directed at improving clearance of plasma cholesterol and preventing atherosclerosis.